Method for treating localized fat deposits

ABSTRACT

The present invention is directed to methods for treating obesity, reducing excessive body weight, treating an obesity-related condition, treating unwanted localized fat deposits, and treating areas of cellulite. The method comprises the steps of first identifying a subject in need thereof, and administering to the subject an effective amount of rhamnolipids. A pharmaceutical composition comprising rhamnolipids can be applied by any accepted mode of administration including oral, intranasal, subcutaneous, percutaneous, intravenous, or intracutaneous administration.

This application is a continuation of U.S. application Ser. No.13/946,931, filed Jul. 19, 2013; which is a continuation ofPCT/US2012/043075, filed Jun. 19, 2012, which claims the benefit of U.S.Provisional Application Nos. 61/499,638, filed Jun. 21, 2011,61/515,806, filed Aug. 5, 2011 and 61/641,117, filed May 1, 2012. Thecontents of the above-identified applications are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to using rhamnolipids for treating obesityand obesity-related conditions such as metabolic syndrome, hypertension,type 2 diabetes, non-alcoholic fatty liver disease or obesity-relatedkidney disease.

BACKGROUND OF THE INVENTION

Obesity is a medical condition in which excess body fat has accumulatedto the extent that it may have an adverse effect on health, leading toreduced life expectancy and/or increased health problems. Body massindex (BMI), a measurement which compares weight and height, definesindividuals as overweight or as suffering from excessive body weight(pre-obese) if their BMI is between 25 and 30 kg/m², and overtly obesewhen their BMI is greater than 30 kg/m². There is increased risk ofco-morbidities for individuals with a BMI between 25.0 to 29.9, andmoderate to severe risk of co-morbidities for individuals with a BMIgreater than 30. Obesity is a serious health and economic burden, andpredisposes an individual to a variety of cardiometabolic diseases.Obesity increases the likelihood of metabolic syndrome, hypertension,type 2 diabetes, non-alcoholic fatty liver disease, or obesity-relatedkidney disease.

Metabolic syndrome is characterized by the presence of three or more ofthe following components: abdominal obesity (waist circumference>102 cmin men, >88 cm in women), elevated triglycerides (>150 mg/dl or on drugtreatment for elevated triglycerides), reduced HDL-C level (<40 mg/dl inmen, <50 mg/dl in women or on drug treatment for reduced HDL-C),hypertension (systolic blood pressure>130 mmHg or diastolic bloodpressure>85 mm Hg or on antihypertensive drug treatment) and impairedfasting glucose (100-125 mg/dl or on anti-diabetic drug treatment).

Hypertension is a chronic medical condition in which the blood pressurein the vasculature is elevated. This requires the heart to work harderthan normal to circulate blood through the blood vessels. Hypertensionis present if it is persistently at or above 140/90 mmHg. According topopulation studies, almost two-thirds of people suffering from obesityare at risk of hypertension.

Diabetes mellitus type 2 (type 2 diabetes) is a metabolic disorder thatis characterized by high blood glucose in the context of insulinresistance and relative insulin deficiency. Obesity is thought to be theprimary cause of type 2 diabetes in those people who are geneticallypredisposed to the disease. Long-term complications from high bloodsugar can include heart disease, strokes, diabetic retinopathy whereeyesight is affected, kidney failure, and poor circulation of limbsleading to amputations.

Non-alcoholic fatty liver disease includes fatty liver (accumulation offat in the liver), non-alcoholic steatohepatitis (fat in the livercausing liver inflammation), and often leads to cirrhosis (irreversible,advanced scarring of the liver as a result of chronic inflammation ofthe liver). All of the stages of non-alcoholic fatty liver disease arenow believed to be due to insulin resistance, a condition closelyassociated with obesity. Moreover, studies demonstrate a correlationbetween BMI and the degree of liver damage in non-alcoholic fatty liverdisease, which shows that the greater the BMI, the greater the liverdamage. Non-alcoholic fatty liver disease is an increasingly commonliver disease in developed countries because of the rising prevalence ofobesity. Elevated transaminases, alanine transaminase (ALT) andaspartate transaminase (AST), are used to monitor non-alcoholic fattyliver disease in obese patients.

Obesity can indirectly influence kidney disease by increasing rates ofdiabetes and hypertension, which are known risk factors for kidneydisease. However, there is growing evidence that obesity per se, even inthe absence of diabetes, significantly increases the risk ofobesity-related kidney disease and adversely impacts its progression.Obesity-related kidney disease is characterized by albuminuria,glomerulomegaly and secondary focal glomerulosclerosis.Glomerulosclerosis refers to a scarring of the kidneys' tiny bloodvessels, the glomeruli, which are the functional units in the kidneythat filter urine from the blood. Weight loss, blockade of the reninangiotensin system, and restoration of adipokine levels may bebeneficial to ameliorate the progression of obesity-related kidneydisease.

Obese Zucker rats (OZRs) have defective brain leptin dependent signaltransduction, resulting in markedly increased food intake and decreasedenergy expenditure. They are used as an animal model of hyperphagia,obesity and associated hyperlipidemia, insulin resistance, fatty liverdisease, and renal disease. OZRs are hyperphagic, hyperlipidemic,hyperinsulinemic, and have severe peripheral insulin resistance,metabolic characteristics also seen in human patients with type 2diabetes and metabolic syndrome. These changes develop during the first20 weeks of life. OZRs typically die of renal failure, specifically froma glomerular disease pathologically similar to human focal segmentalglomerulosclerosis. Various experimental maneuvers have been found toattenuate the development of glomerular disease in OZRs, includingtreatment with lipid-lowering agents, ovariectomy, and reduction of foodintake (Stevenson, F. T., et al., Obesity Research (2001) 9:492-499;Koteish, A., Diehl, A. M., Liver Dis (2001) 21:89-104).

Obesity is often accompanied by excess fat storage in tissues other thanadipose tissue, including liver and skeletal muscle, which may lead tolocal insulin resistance and may stimulate inflammation, as insteatohepatitis. In addition, obesity changes the morphology andcomposition of adipose tissue, leading to changes in protein productionand secretion. Some of these secreted proteins, including severalpro-inflammatory mediators, may be produced by macrophages resident inthe adipose tissue. The changes in inflammatory status of adipose tissueand liver with obesity feed a growing recognition that obesityrepresents a state of chronic low-level inflammation.

Adipose tissue has a primary role in the pathogenesis of non-alcoholicfatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH),namely the central (or upper body) obesity phenotype associated withincreased visceral fat. Studies have shown that the amount ofintra-hepatocellular lipids increases by approximately 20% for any 1%increase in total or subcutaneous adipose tissue, but doubles for any 1%increase in intra-abdominal adipose tissue. Therefore, even modestincreases in visceral fat (in the absence of increased body mass index(BMI)) may cause steatosis (Bugianesi E., et al., Diabetologia (2005)48:634-642).

Lipomas are adipose tissue tumors, which are benign, slow-growing tumorscomposed of enlarged adipose tissue cells, preferentially insubcutaneous tissue. They can become painful and the compression derivedtherefrom on blood vessels may cause neuralgia. Subcutaneousaccumulations of fat or proliferations of adipose cells such as lipomasor lipedemas are predominantly treated by surgical means throughliposuction or direct surgical removal. Treatment measures of thesetypes are associated with the known complications or risks caused byanesthesia, local reactions and possible infections, and in somecircumstances, require admission to a hospital ward. There are currentlyno FDA-approved injections for the reduction or elimination of localunwanted body fat.

Cellulite is a skin alteration often described as an “orange peel,”“mattress” or “dimpling” appearance on the thighs, buttocks andsometimes lower abdomen and upper arms of otherwise healthy women.Cellulite is caused by small protrusions of fat called papillae adiposaeinto the dermis. This structural alteration of subcutaneous fatprotruding (or herniating) into the dermis gives skin the bumpyappearance referred to as cellulite. Individuals with cellulite andhigher BMIs have a weaker, less dense connective tissue structure,leading to increased extrusion of adipose tissue lobules through thehypodermis. These individuals have a higher amount of extrusion ofadipose tissue while the thickness of the dermis is significantly lower.Affected individuals with lower BMIs show differences in the thicknessof the adipose tissue layer, with a significantly thicker adipose layerin individuals with clinically evident cellulite.

SUMMARY OF THE INVENTION

The present invention is directed to a method for reducing excessivebody weight from a subject. The method comprises the steps ofidentifying a subject suffering from excessive weight or overt obesity,and administering to the subject an effective amount of rhamnolipids.

The present invention is also directed to a method for treating anobesity-related condition in a subject. The method comprises the stepsof first identifying a subject suffering from (i) excessive body weightor overt obesity, and (ii) an obesity-related condition, andadministering to the subject an effective amount of rhamnolipid, whereinsaid obesity-related condition is metabolic syndrome, hypertension, type2 diabetes, non-alcoholic fatty liver disease, or obesity-related kidneydisease.

The present invention is further directed to a method for treatingunwanted localized fat deposits in a subject. The method comprises thesteps of first identifying a subject suffering from localized fatdeposits and administering into or around the areas of localized fatdeposits of the subject an effective amount of rhamnolipids, whereby thelocal fat deposits are reduced.

The present invention is further directed to a method for treating areasof cellulite in a human subject. The method comprises the steps of firstidentifying a human subject suffering from cellulite and administeringinto or around the areas of cellulite an effective amount ofrhamnolipids, whereby the appearance of cellulite is improved.

The pharmaceutical composition comprising the active compound can beapplied by any accepted mode of administration including oral,intranasal, subcutaneous, percutaneous, intravenous, or intracutaneousadministration. Subcutaneous administration is preferred.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the plotted ANOVA results from an analysis comparing theproportion of the difference of post-treatment to pre-treatment bodyweights of groups of mice treated with RPI-100, RPI-200, RPI-300, andthe placebo. Error bars represent the 95% confidence intervals for eachgroup.

FIG. 2 is a mirror image photograph of the subject illustrating thevisual decrease in localized fat observed following subcutaneousinjections with rhamnolipids. The white arrow on the left indicates thesubject's left iliac region injection site and the white arrow on theright indicates the subject's right lumbar region of anterior abdomeninjection site.

FIG. 3 shows the mean body weight (grams) of the rats treated withRPI-100 or the placebo. Body weight was measured on each day of thestudy (Study Day). Mean body weights were calculated and plotted for thegroups treated with the placebo (black line) or RPI-100 (black dots).Rats were treated with either RPI-100 or the placebo on Study Days 0, 7,9, 12, 13, 14, 19, 23, 26, 28, and 30 (Dosed).

FIG. 4 shows mean blood glucose levels (SEM) of the placebo (grey bar)and RPI-100 (white bar) treated rats.

FIG. 5 shows mean cholesterol (left) and triglyceride (right) levels ofthe placebo (grey bar) and RPI-100 (white bar) treated rats.

FIG. 6 shows the plotted mean body weight (grams) of the rats treatedwith RPI-100 or the placebo. Rats were treated with either RPI-100 orthe placebo three times per week. Body weight was measured on each dayof dose administration. Mean body weights were calculated and plottedfor the groups treated with the placebo (black line) or RPI-100 (blackdots). Rats were fasted from Day 14-15.

FIG. 7 shows average daily food consumption for the placebo (grey bars)and RPI-100 (white bars) treated rats. The average daily foodconsumption was calculated by measuring the total amount of foodconsumed (grams) per rat during a specified time period (Day 1-4, Day4-8, Day 18-23, Day 23-25).

FIG. 8 shows mean alanine aminotransaminase (ALT) levels (U/L of theplacebo (grey bar) and RPI-100 (white bar) treated rats.

FIG. 9 shows mean triglyceride levels (MG/DL) of the placebo (grey bar)and RPI-100 (white bar) treated rats.

FIG. 10 shows mean creatinine levels (MG/DL) of the placebo (grey bar)and RPI-100 (white bar) treated rats.

Error bars in FIGS. 3-6 and 8-10 represent the 95% confidence intervalsfor each group.

DETAILED DESCRIPTION OF THE INVENTION

The inventor has discovered that rhamnolipids are effective for reducingexcessive body weight, treating obesity-related conditions such asmetabolic syndrome, hypertension, type 2 diabetes, non-alcohol fattyliver disease, or obesity-related kidney disease, removing unwantedlocalized fat deposits, and treating cellulite areas in a subject. Byadministering rhamnolipids to a subject, the subject's condition ordisorder is improved.

Rhamnolipids

Rhamnolipids are biosurfactants containing rhamnose sugar molecules andβ-hydroxyalkanoic acids. Rhamnolipids suitable to be used in the presentinvention include natural rhamnolipids, for example, obtained fromPseudomonas aeruginosa; rhamnolipids produced by any Pseudomonad,including P. chlororaphis, Burkholdera pseudomallei, Burkholdera(Pseudomonas) plantarii, and any recombinant Pseudomonad. Suitablerhamnolipids also include those produced by other bacteria or by plantseither naturally or through (genetic) manipulation. Suitablerhamnolipids further include rhamnolipids and their analogs prepared bychemical synthesis or expression by mammalian cells. Suitablerhamnolipids include those disclosed in U.S. Pat. Nos. 7,262,171 and5,514,661, in which the structures of rhamnolipids are incorporatedherein by reference.

Suitable rhamnolipid formulations contain one or more rhamnolipids offormula (I)

wherein:R¹═H, unsubstituted α-L-rhamnopyranosyl, α-L-rhamnopyranosyl substitutedat the 2 position with a group of formula —O—C(═O)—CH═CH—R⁵, or—O—C(═O)—CH═CH—R⁵;R²═H, C₁₋₆alkyl, —CHR⁴—CH₂—COOH or —CHR⁴—CH₂—COOR⁶; andR³-R⁶ are independently alkyl.“Alkyl” refers to groups of from 1 to 12 carbon atoms, either straightchained or branched, preferably from 1 to 8 carbon atoms, and morepreferably 1 to 6 carbon atoms.In one embodiment, R³=—(CH₂)_(x)—CH₃, wherein x=4-19;R⁴=—(CH₂)_(y)—CH₃, wherein y=1-19;R⁵=—(CH₂)_(z)—CH₃, wherein z=1-12; andR⁶=—C₁₋₆alkyl,

Useful rhamnolipids of the Formula 1 includeα-rhamnopyranosyl-(1,2)-α-L-ramnopyranosyl)-3-hydroxydecanoyl-3-hydroxsydecanoicacid and has the following structure (Formula 2):

In one embodiment, R¹=α-L-rhamnopyranosyl substituted at the 2-positionby —O—C(═O)—CH═CH—R⁵, R²=—CHR⁴—CH₂—COOH, R³=—(CH₂)₅—CH₃, R⁴=—(CH₂)₂—CH₃,and R⁵=—(CH₂)₅—CH₃.

In another embodiment, R¹=α-L-rhamnopyranosyl substituted at the2-position by —O—C(═O)—CH═CH—R⁵, R²=—CHR⁴—CH₂—COOH, R³=—(CH₂)₅—CH₃,R⁴=—(CH₂)₅—CH₃, and R⁵=—(CH₂)₅—CH₃;

In another embodiment, R¹=α-L-rhamnopyranosyl substituted at the2-position by —O—C(═O)—CH═CH—R⁵, R²=—CHR⁴—CH₂—COOCH₃, R³=—(CH₂)₅—CH₃,R⁴=—(CH₂)₂—CH₃, and R⁵=—(CH₂)₅—CH₃.

Preferred rhamnolipids areL-rhamnosyl-β-hydroxydecanoyl-β-hydrocydecanoate (rhamnolipids 1,Rha-C10-C10) andL-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate(rhamnolipids 2, Rha-Rha-C10-C10), and the mixture thereof.

Some common di-rhamnolipids useful for this invention include:L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydodecanoate(often referred to as Rha-Rha-C10-C12);L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxytetradecanoyl-beta-hydroxytetradecanoate(often referred to as Rha-Rha-C14-C14).

Pharmaceutical Compositions

One or more rhamnolipids, which are the active ingredient of the presentinvention, can be used directly as a pharmaceutical composition. One ormore rhamnolipids can also be formulated in a pharmaceutical compositionwhich comprises rhamnolipids and one or more pharmaceutically acceptablecarriers. The pharmaceutical composition can be in the form of a liquid,a solid, or a semi-solid.

Pharmaceutically acceptable carriers can be selected by those skilled inthe art using conventional criteria. Pharmaceutically acceptablecarriers include, but are not limited to, sterile water or salinesolution, aqueous electrolyte solutions, isotonicity modifiers, waterpolyethers such as polyethylene glycol, polyvinyls such as polyvinylalcohol and povidone, cellulose derivatives such as methylcellulose andhydroxypropyl methylcellulose, polymers of acrylic acid such ascarboxypolymethylene gel, nanoparticles, polysaccharides such asdextrans, and glycosaminoglycans such as sodium hyaluronate and saltssuch as sodium chloride and potassium chloride.

In one embodiment, the pharmaceutical composition containsrhamnolipid(s) and a pharmaceutically acceptable carrier in a liquidform. The concentration of rhamnolipid(s) in a liquid pharmaceuticalcomposition is in general about 0.005-20% (w/w), or 0.02-5%, or 0.1-5%,or 0.2 to 5%, or 0.2-2%, or 0.05-0.5% (w/w). In one embodiment, theconcentration of rhamnolipid(s) is about 0.2-2%, for example, 0.5%(w/w). In another embodiment, the concentration is about 10-20%, forexample, about 15%. “About” as used herein, refers to ±10% of therecited value. The concentration can be higher or lower as long as itdelivers an effective and tolerable dose per dosing interval.

In one embodiment, the pharmaceutical composition of the presentinvention provides an aqueous solution comprising water andrhamnolipids; the composition optionally comprises suitable ionic ornon-ionic tonicity modifiers, suitable buffering agents, andrhamnolipids. In one embodiment, the rhamnolipid is at 0.005-20% (w/w),and the aqueous solution has a tonicity of 200-400 mOsm/kG and a pH of4-9.

The pharmaceutical composition is preferably formulated to have a pHbetween 4.5-8, more preferably 5-7.4. The pharmaceutical composition mayoptionally contain a buffer to facilitate a stable pH of 5-7.4.

The pharmaceutical composition optionally contains non-ionic tonicityagents such as mannitol, sucrose, dextrose, glycerol, polyethyleneglycol, propylene glycol, or ionic tonicity agent such as sodiumchloride. The pharmaceutical composition can further contain ionic ornon-ionic surfactants, bile salts, phospholipids, cyclodextrins,micelles, liposomes, emulsions, polymeric microspheres, nanoparticles,other biodegradable microsphere technology, or their combination.

In one embodiment, the pharmaceutical composition is in a dosage formsuch as tablets, capsules, granules, fine granules, powders, syrups,suppositories, injectable solutions, or the like. The abovepharmaceutical composition can be prepared by conventional methods.

For example, a tablet formulation or a capsule formulation may containother excipients that have no bioactivity and no reaction withrhamnolipids. Excipients of a tablet may include fillers, binders,lubricants and glidants, disintegrators, wetting agents, and releaserate modifiers. Binders promote the adhesion of particles of theformulation and are important for a tablet formulation. Examples ofbinders include, but not limited to, carboxymethylcellulose, cellulose,ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karayagum, starch, starch, and tragacanth gum, poly(acrylic acid), andpolyvinylpyrrolidone. A tablet formulation may contain 1-90% ofrhamnolipids. A capsule formulation may contain 1-100% of rhamnolipids.

In one embodiment, the pharmaceutical composition comprises one or morerhamnolipids, a serum albumin, and a pharmaceutically acceptablecarrier. In this pharmaceutical composition, a majority of therhamnolipids are non-covalently bound to the fatty acid binding sites ofalbumin or to other binding sites of albumin. Albumin bound rhamnolipidsprolong the beneficial effects of rhamnolipids so that longer dosingintervals can be used.

In another embodiment, the pharmaceutical composition comprises one ormore rhamnolipids imbedded in a solid or semi-solid matrix, and is in aliquid, solid, or semi-solid form. The pharmaceutical composition can beinjected subcutaneously to a subject and then the active ingredientsslowly released in the subject. The formulation may contain 1-90%rhamnolipids.

The pharmaceutical composition is preferred to be stable at roomtemperature for at least 6 months, 12 months, preferably 24 months, andmore preferably 36 months. Stability, as used herein, means thatrhamnolipid maintains at least 80%, preferably 85%, 90%, or 95% of itsinitial activity value.

The pharmaceutical compositions of the present invention can be preparedby aseptic technique. The purity levels of all materials used in thepreparation preferably exceed 90%.

Methods of Use

The present invention is directed to a method for reducing excessivebody weight from a subject. The method comprises the steps ofidentifying a subject suffering from excessive weight or overt obesity,and administering to the subject an effective amount of rhamnolipids. Ahuman subject suffering from excessive body weight (pre-obese) isdefined by a Body Mass Index (BMI) between 25 and 30 kg/m² and a humansubject suffering from overt obesity is defined by BMI greater than 30kg/m². Rhamnolipids effectively reduce excessive body weight of apre-obese subject or an overtly obese subject.

The present invention is also directed to a method for treating obesity.The method comprises the steps of identifying a subject suffering fromovert obesity, and administering to the subject an effective amount ofrhamnolipids, whereby the excessive body weight of the subject isreduced.

The present invention is directed to a method for treating anobesity-related condition in a subject. The method comprises the stepsof first identifying a subject suffering from (i) excessive body weightor overt obesity, and (ii) an obesity-related condition, andadministering to the subject an effective amount of rhamnolipid, whereinsaid obesity-related condition is metabolic syndrome, hypertension, type2 diabetes, non-alcoholic fatty liver disease, or obesity-related kidneydisease.

In one embodiment, the method reduces or alleviates the symptoms of anobesity-related condition, metabolic syndrome. The present inventionprovides a method to treat metabolic syndrome characterized by centralobesity; fasting hyperglycemia, which includes type 2 diabetes, impairedglucose tolerance or insulin resistance; elevated blood pressure,triglyceride, cholesterol, and blood glucose levels. The presentinvention is effective in reducing body weight, blood pressure, andtriglyceride, cholesterol, and blood glucose levels.

In another embodiment, the method reduces or alleviates the symptoms ofan obesity-related condition, hypertension. The present inventionprovides a method to treat hypertension characterized by truncal obesityand elevated blood pressure. The present method reduces elevated bloodpressure.

In another embodiment, the method reduces or alleviates the symptoms ofan obesity-related condition, type 2 diabetes. The present inventionprovides a method to treat type 2 diabetes characterized by insulinresistance, where the fat, liver, and muscle cells do not respondcorrectly to insulin, and as a result, blood sugar does not get storedin these cells for energy. Type 2 diabetes is most often characterizedby a BMI>25 kg/m² and increased levels of blood glucose compared withnormal subjects. The present invention effectively reduces body weightand blood glucose levels in subjects with type 2 diabetes.

In another embodiment, the method reduces or alleviates the symptoms ofan obesity-related condition, non-alcoholic fatty liver disease. Thepresent invention provides a method to treat non-alcoholic fatty liverdisease characterized by fat deposited in the liver not due to excessivealcohol use, obesity and elevated liver transaminase levels,specifically alanine aminotransaminases (ALT). The present invention iseffective in decreasing body weight and liver transaminase (ALT) levels.

In another embodiment, the method reduces or alleviates the symptoms ofan obesity-related condition, obesity-related kidney disease. Thepresent invention provides a method to treat obesity-related kidneydisease characterized by damage to the renal glomeruli and an increasein creatinine levels. For example, the method is effective to treatglomerulosclerosis, which is characterized by scarring of the kidney'sglomeruli, obesity, and elevated creatinine levels. The presentinvention effectively reduces body weight and creatinine levels.

The present invention is directed to a method for treating unwantedlocalized fat deposits in a subject. The method comprises the steps offirst identifying a subject suffering from localized fat deposits andadministering into or around the areas of localized fat deposits of thesubject an effective amount of rhamnolipids, whereby the local fatdeposits are reduced.

In one embodiment, the method reduces unwanted localized fat deposits.The present invention provides a method to treat unwanted localized fatdeposits characterized by excess subcutaneous adipose tissue. Thepresent invention effectively reduces unwanted subcutaneous adiposetissue.

In one embodiment, the method reduces unwanted localized fat from asubject suffering from lipodystrophy, which is characterized by abnormalconditions of the subject's adipose tissue. Examples of lipodystrophyinclude, but are not limited to lipodystrophy caused by metabolicabnormalities due to genetic issues, which are characterized by insulinresistance and are associated with metabolic syndrome; andHIV-associated lipodystrophy, as characterized by the presence of anenlarged dorsocervical fat pad, circumferential expansion of the neck,breast enlargement, and/or abdominal visceral fat accumulation.

In another embodiment, the method reduces unwanted localized fat from asubject suffering from lipoma, which is characterized by a benign tumorof adipose subcutaneous tissue.

The present invention is directed to a method for treating areas ofcellulite in a human subject. The method comprises the steps of firstidentifying a human subject suffering from cellulite and administeringinto or around the areas of cellulite an effective amount ofrhamnolipids, whereby the appearance of cellulite is improved. Thepresent invention provides a method to treat cellulite characterized bya modification of skin topography evident by skin dimpling andnodularity that is caused by the protrusion of subcutaneous fat withinfibrous connective tissue, leading to a padded or orange peel-likeappearance.

In the above treatment methods, rhamnolipids can be the only activeingredient in a treatment method, or rhamnolipids can be used togetherwith another active ingredient that is useful for the treatment.

“An effective amount,” as referred to in the above methods, is theamount effective to treat a disease by ameliorating the condition orreducing the symptoms of the disease.

The pharmaceutical composition of the present invention can be appliedby systemic administration. Systemic administration includes oral,intranasal, subcutaneous, percutaneous, or intravenous administration,and other systemic routes of administration, e.g., intracutaneousinjection. Subcutaneous administration is the preferred route ofadministration for the present invention. Intracutaneous injection canbe used for reducing areas of cellulite.

In one embodiment, the pharmaceutical composition is applied 1-3 timesdaily, 1-2 times daily, once daily, once every 2-3 days, once weekly,once every 2 weeks, once every 4 weeks, or once every one to threemonths by intranasal, intravenous, subcutaneous, percutaneous, orintracutaneous administration.

In one embodiment, the pharmaceutical composition is an oral formulationthat is taken 1-4 times daily, once every 2-3 days, once weekly, onceevery 2 weeks, or once every 4 weeks.

In another embodiment, the pharmaceutical composition is a delayedrelease formulation injected intracutaneously.

Those of skill in the art will recognize that a wide variety of deliverymechanisms are also suitable for the present invention.

The present invention is useful in treating a mammalian subject, such ashumans, dogs and cats. The present invention is particularly useful intreating humans.

The following examples further illustrate the present invention. Theseexamples are intended merely to be illustrative of the present inventionand are not to be construed as being limiting.

EXAMPLES Example 1 Objective

Originally, the objective of this study was to determine the effects oftreatment with three different pharmaceutical formulations ofrhamnolipids within the context of an allergic rhinitis study in mice.However, the weight loss effects due to treatment with the variousrhamnolipids were unexpectedly discovered during the course of thisstudy.

Materials Animal:

Balb/C mice were used in this study and were eight weeks old. Thetreatment groups and the placebo group were each composed of 10 mice.Allergenization was performed in a similar fashion as reported in theliterature.

Sensitization Materials:

The following were used to sensitize and induce allergic rhinitis in themice:

1. OVA/Alum (0.8 μg and 0.8 mg/mouse, respectively) was used as anintraperitoneal injection. Grade V OVA was from Sigma-Aldrich (St.Louis, Mo.). Alum was aluminum hydroxide gel from Pierce Biotechnology(Rockford, Ill.); now Thermo Fisher Scientific, Inc.)

2. An ovalbumin (OVA) solution (5 μL of 50 mg/ml of stock OVA solutionin 1×PBS) was administered intranasally.

Rhamnolipid Mixtures:

1. RPI-100: JBL515 was supplied by Jeneil Biotech (Saukville, Wis.) as a15% solution of a rhamnolipid mixture of a combination ofmono-Rhamnolipid (Rha-C₁₀-C₁₀) and di-Rhamnolipid (Rha-Rha-C₁₀-C₁₀)consisting at a ratio of approximately 1:1.21, respectively.

The 15% solution was diluted in 0.95% saline to prepare a dosingsolution of 0.5%.

2. RPI-200: JBR8% was supplied by Jeneil Biotech (Saukville, Wis.) as an8% solution of a mono-Rhamnolipid (Rha-C₁₀-C₁₀).

The 8% solution was diluted in 0.95% saline to prepare a dosing solutionof 0.5%.

3. RPI-300: KIT was supplied by Karlsruher Institut für Technologie(KIT) (Karlsruhe, Germany) as a powder of di-Rhamnolipid(Rha-Rha-C₁₀-C₁₀).

The powder was reconstituted in 0.95% saline (5 mg/ml) to prepare adosing solution of 0.5%.

Saline was used as the placebo in this study.

Methods

Mice were sensitized once-weekly with four intraperitoneal injections ofOVA/Alum (Day 0, 5, 14 and 21). From Day 22-39, anesthetized mice wereadministered once-daily doses of the OVA stock solution instilled ineach nostril.

On Day 37, mice were assigned to one of three rhamnolipid treatmentgroups or a placebo group. The rhamnolipid treatment groups received arhamnolipid mixture containing RPI-100, RPI-200, or RPI-300. The mice ineach treatment group received 5 μl of the designated 0.5% rhamnolipidmixture in saline per nostril at two different time points on Day 37,for a total applied volume of 20 μl. Whereas, the mice in the placebogroup received 5 μl of normal saline per nostril at the same twodifferent time points on Day 37, for a total applied volume of 20 μl.The doses were administered 3 hours and 2 hours prior to the intranasaldose of the OVA solution. Body weights of the mice were measured andrecorded on Days 35, 37, and 39 as part of a standard animal safetyobservation protocol.

Results

Post-Treatment with Intranasal Doses of Rhamnolipids

Gross body weight differences were observed 2 days post-treatment (Day39) when compared with 2 days pre-treatment (Day 35) body weightmeasurements. A formal statistical analysis was performed using ananalysis of variance (ANOVA) (www.physics.csbsju.edu) to compare theproportion of the difference of post-treatment to pre-treatment bodyweights of the respective treatment groups and the placebo group. Allfour groups, including the groups of mice treated with RPI-100, RPI-200,RPI-300, and the placebo group, were included in the ANOVA. Theprobability of the ANOVA result, assuming the null hypothesis, was lessthan 0.0001. The data were presented graphically with error barsrepresenting the 95% confidence intervals (CI) of each group (FIG. 1).Thus, these results show that there was a statistically significantreduction in body weight in the mice treated with rhamnolipids whencompared to those mice treated with the placebo.

Conclusions:

The results of this study demonstrate that intranasal administration ofthree different formulations of rhamnolipids was effective in elicitingweight loss in normal weight mice. Specifically, treatment of mice withmono-rhamnolipid, di-rhamnolipid, or a combination of bothmono-rhamnolipid and di-rhamnolipid resulted in a statisticallysignificant reduction of body weight when compared to those mice treatedwith the placebo.

Example 2 Objective

The objective of this study was to explore the effects and feasibilityof subcutaneous injections of rhamnolipids at varying concentrations ina human model.

Materials Subject:

Human subject: 60 year old male, 108 kg (BMI 29.8) with knownhypertension.

Injections:

Standard insulin syringes (Monoject®) were used for injections.

Rhamnolipids:

15% (w/v) solution of rhamnolipid: JBL515 was used for the 15% solutionof rhamnolipid. 1, 2.5, 5, and 10% (w/v) solution of rhamnolipid: Eachsolution was prepared by diluting JBL515 (15%) in water.

Methods and Results Study Part A: Initial Study of Small, IncreasingDoses of Rhamnolipid Injections

The subject injected himself with initial small test doses ofapproximately 0.5 ml of a 1% solution of rhamnolipids into subcutaneousfatty tissue located in his left lumbar region and his right iliacregion.

As these initial injections were extremely well tolerated with no signsof significant inflammation, a subsequent test was performed todetermine the potential effects of increasing concentrations ofrhamnolipid injections. The subject injected himself with incrementallyincreasing concentrations of rhamnolipid injections into thesubcutaneous area of his right lumbar region of his anterior abdomen andleft iliac region every other day beginning on Day 1 and ending on Day 9(Table 1). A transient hardening of the injected subcutaneous fattytissue along with a subsequent reduction in overall volume of theunderlying fatty tissues was observed over the next 10 dayspost-injection.

TABLE 1 Study Part A: Administration of Increasing Concentrations ofRhamnolipids Day 1 Day 3 Day 5 Day 7 Day 9 % Solution of 1% 2.5% 5% 10%15% rhamnolipid # of injections 1 2 2 2 2 Volume of 0.5 ml 0.5 ml 0.5 ml0.5 ml 0.5 ml each injection Total volume 0.5 ml of 1% 1.0 ml of 2.5%1.0 ml of 5% 1.0 ml of 10% 1.0 ml of 15% and % of rhamnolipidadministered per day

Study Part B: Exposure to Multiple Subcutaneous Rhamnolipid Injections

One week after completion of Study Part A, the subject injected himselfwith a total of 6 injections of 0.5 ml of a 15% solution ofrhamnolipids. These injections were placed into the subcutaneous fattissues of the subject's right lumbar region of his abdomen and his leftiliac region on Day 1 of Study Part B. Each injection site was separatedby approximately 1-2 inches. Over the next 10 days post-injection, ahardening of the injected subcutaneous fatty tissue along with asubsequent reduction in overall volume of the underlying fatty tissueswas observed.

During this study, the subject showed a reduction in blood pressure.Prior to the rhamnolipid injections, the subject's typical bloodpressure was, on average, approximately 135/85 mmHg (systolic/diastolic)while taking his prescribed anti-hypertensive medications (once daily 50mg metoprolol and once daily 25 mg hydrochlorothiazide). However, on Day3, the subject's blood pressure was reduced to approximately 110/65 mmHgwhile the subject was still taking his anti-hypertensive medications asprescribed. On Day 4, the subject discontinued taking his metoprololmedication, and his blood pressure remained, on average, around 110/65mmHg. On Day 5, the subject discontinued taking his hydrochlorothiazidemedication. On Day 8, the subject injected himself with an additionalseries of 6 injections of 0.5 ml of a 15% solution of rhamnolipids intothe subcutaneous fat tissues of his right lumbar region of his anteriorabdomen and his left iliac region. The subject's blood pressure remainedwithin a range of 110/67 mmHg to 132/80 mmHg from Day 8 to Day 14. OnDay 15, the subject's blood pressure started to rise to values of 145/82mmHg to 155/85 mmHg, which were similar to the values seen prior totreatment.

Additionally, over the course of these studies, which lastedapproximately 8 weeks, the subject's body weight decreased from 108 to98.5 kg (BMI 27.1). There were no dietary restrictions for the subjectduring this study.

A mirror image photograph of the subject illustrates the exemplaryresults achieved in this study (FIG. 2). The white arrows located on theleft and right side of the photograph identify the injection sites onthe subject's left iliac region and right lumbar region of his anteriorabdomen. No scarring, skin deformation, or painful knots under the skinwas observed at any of the subject's injection sites.

Conclusion

The results of this study demonstrate that subcutaneous injections withrhamnolipids, specifically, a combination of mono-rhamnolipid anddi-rhamnolipid, resulted in a visual reduction of fat deposits andoverall body weight in a human subject. These results provide evidenceto suggest that rhamnolipids can be used for the treatment of unwantedlocalized fat deposits in patients. Additionally, the reduction ofsubcutaneous fatty tissue at the rhamnolipid injection sites accompaniedby local tolerability suggests that the increased intracutaneous fatprotrusion observed in cellulite can be reduced through intracutaneousand subcutaneous local injections of rhamnolipids. Furthermore, theconcurrent decrease seen in the subject's blood pressure suggests thatrhamnolipids can be used to treat hypertension.

Example 3 Objective

The objective of this study was to determine the effects of differenttreatment intervals and prolonged exposure of rhamnolipids on bodyweight and parameters of metabolic syndrome in rats.

Materials Animal:

Five obese Zucker rats (Charles River Laboratories International, Inc.(Wilmington, Mass.), approximately 8 weeks old, were used in this study.

Rhamnolipids:

RPI-100 was used in this study.

Saline was used as a placebo for this study.

Methods

Three rats were treated with interval, subcutaneous injections ofRPI-100, while two rats were injected with a placebo. Fixed doses (120mg/kg body weight) were administered as two 60 mg/kg injections intodorsal fat regions of the rats on a total of 11 days (Study Day 0, 7, 9,12, 13, 14, 19, 23, 26, 28, 30) (FIG. 3). The sites of injections werealternated with each dose administration. Blood glucose, cholesterol,and triglyceride levels were measured on Day 39, which was 9 days afterthe last injection of RPI-100 (FIGS. 4-5). Renal biopsies were takenupon necropsy on Day 39 and histology findings analyzed (Table 2). Allclinical assessments were performed by Quality Veterinary Laboratory,LLC. (Davis, Calif.). Animals were not fasted prior to clinicallaboratory and necropsy assessments. Body weights of the rats weremeasured at the beginning of each day and recorded (FIG. 3).

Results

As demonstrated in FIG. 3, subcutaneous injections with RPI-100 wereassociated with a significant decrease in body weight gain on Day 35 (5days after the last day of dosing) (two-tailed P value=0.0480 (unpairedt-test)). Additionally, rats treated with RPI-100 showed a decrease inblood glucose, triglyceride and cholesterol levels on Day 39 (9 daysafter the last day of dosing) compared with rats that were injected withthe placebo (FIGS. 4-5). The graph presented in FIG. 4 indicates thatthe differences seen in blood glucose levels between the RPI-100 andplacebo group are statistically significant because the 95% CI bars fromthe rat group treated with RPI-100 do not overlap with the bars from thegroup treated with the placebo (P value=0.0349 (unpaired t-test)).Finally, the rats that received the placebo presented with initialhistological signs consistent with diabetic renal damage, whereas therats that received the RPI-100 did not show significant lesions (Table2).

TABLE 2 Renal Histology Group Animal Kidney Histology Placebo 101 Renaltubular dilation, mild, multifocal 102 Renal tubular dilation andbasophilia, moderate, multifocal RPI-100 201 NSL (No significantlesions) 202 NSL 203 NSL

Conclusion

This pilot study demonstrates that interval injections with RPI-100 in arat model were associated with a statistically significant difference inbody weight gain. This result was concomitant with improvements inparameters of metabolic syndrome, such as blood glucose, cholesterol,and triglyceride levels compared to rats that received the placebo.These results suggest that rhamnolipids can be used for the treatment ofmetabolic syndrome and type 2 diabetes. In addition, the rats thatreceived the placebo presented with initial histological signs of renaldamage in contrast to the rats that received RPI-100, which suggeststhat rhamnolipids can be used to treat type 2 diabetes, obesity-relatedkidney disease, and glomerulosclerosis.

Example 4 Objective

The objective of this study was to use a larger population of rats toconfirm the consistent and rapid reduction of body weight gain observedupon treatment with rhamnolipids as seen in the pilot study presented inExample 3, and to further assess effects on parameters of metabolicsyndrome, non-alcoholic fatty liver disease and renal disease.

Materials Animals:

Fifteen obese Zucker rats (Harlan Laboratories (Livermore, Calif.)),approximately 8 weeks old, were used in this study.

Rhamnolipids:

RPI-100 was used in this study.

Saline was used as a placebo in this study.

Methods

In this study, seven rats received RPI-100 and eight rats received theplacebo. Fixed doses (120 mg/kg body weight) of RPI-100 wereadministered as two 60 mg/kg subcutaneous injections into dorsal fatregions of the rats. Injections were administered three days a week for4 weeks (Days 0-27) (FIG. 6). The injection sites were alternated witheach dose administration. Clinical laboratory assessments were performedon the third day after the last dose administration (Day 30) (FIGS.8-10). Animals were provided ad libetum access to chow, except on Days14-15 when they were fasted. Animals were not fasted prior to clinicalsafety and hematological laboratory assessments, which were performed onDay 30. Body weights of the rats were measured and recorded on each daythat RPI-100 was administered (FIG. 6). All clinical assessments wereperformed by Quality Veterinary Laboratory, LLC. (Davis, Calif.).

Results

Rats that were administered interval injections with RPI-100 showed astatistically significant difference in body weight from Day 9 throughDay 27 (last day of dosing) (FIG. 6). The P values were <0.05 for theunpaired t-tests comparing body weight differences between RPI-100 andplacebo treated groups for Study Days 9 through 27 (Table 3).

TABLE 3 P-Values (Unpaired T-Tests) Comparing Body Weight Differencesover Time for RPI-100 and Placebo Treated Groups Study Day P value Day 90.0137 Day 11 0.0056 Day 14 0.0027 Day 16 0.0018 Day 17 0.0003 Day 190.0005 Day 21 0.0004 Day 23 0.0003 Day 25 0.0006 Day 26 0.0021 Day 270.0009

Additionally, average food consumption of the rats treated with RPI-100was reduced compared with placebo treated rats (FIG. 7). A statisticallysignificant difference was observed in total food consumption measuredbetween Days 1-25 (P=0.0407 (unpaired t-test)).

Liver transaminase levels, specifically alanine aminotransaminases(ALT), are indicators of non-alcoholic fatty liver disease and weredecreased (improved) in animals treated with RPI-100 (FIG. 8) (ALT Pvalue=0.0261 (unpaired t-test)). Triglyceride values, excluding oneoutlier with the greatest value from each treatment group, weresignificantly decreased in the RPI-100 treated rats compared with theplacebo treated rats (FIG. 9) (triglyceride P value=0.0228 (unpairedt-test)). Finally, creatinine levels, which are an indicator of renalfunction that increase during renal failure, were significantly reducedin animals treated with RPI-100 as indicated by the 95% CI bars (FIG.10) (creatinine P value=0.0285 (unpaired t-test)). No significantdifferences in red or white blood cell counts, or hematocrit wereobserved.

Conclusion

The results in this study showed a significant reduction of body weightgain upon treatment with RPI-100 in obese Zucker rats, which indicatesthat rhamnolipids can be used for treating obesity. Additionally, thisstudy also demonstrates that rhamnolipids can be used to treat metabolicsyndrome because the rats treated with RPI-100 showed significantimprovements in triglyceride levels. These favorable effects onmetabolic syndrome are further supported by the observations seen inprevious examples showing that blood pressure was improved in apre-obese individual (Example 2) and that blood glucose levels weresignificantly reduced in obese Zucker rats (Example 3) followingtreatment with rhamnolipids.

Obese Zucker rats are predisposed to developing non-alcoholic fattyliver disease and obesity-related kidney disease, and ALT levels are animportant parameter in non-alcoholic fatty liver disease. Therefore, thedecrease seen in ALT values in obese Zucker rats treated with RPI-100shows that rhamnolipids can be of utility to treat non-alcoholic fattyliver disease and non-alcoholic steatohepatitis (NASH). Finally, ascreatinine levels are an important parameter in obesity-related kidneydisease, the results demonstrate that rhamnolipids can also be used totreat obesity-related kidney disease and glomerulosclerosis because ratstreated with RPI-100 showed a statistically significant decrease increatinine values compared with those that received the placebo.

The invention, and the manner and process of making and using it, arenow described in such full, clear, concise and exact terms as to enableany person skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the scope of the present invention as setforth in the claims. To particularly point out and distinctly claim thesubject matter regarded as invention, the following claims conclude thespecification.

1-5. (canceled)
 6. A method for treating unwanted localized fat depositsin a mammal subject, comprising the steps of: identifying a subjectsuffering from localized fat deposits, and administering into or aroundthe areas of localized fat deposits of the subject an effective amountof rhamnolipids, whereby the local fat deposits are reduced.
 7. Themethod according to claim 6, wherein said subject suffers fromlipodystrophy or lipoma.
 8. The method according to claim 6, whereinsaid rhamnolipids are compounds of Formula (I),

wherein: R¹═H, unsubstituted α-L-rhamnopyranosyl, or α-L-rhamnopyranosylsubstituted at the 2 position with —O—C(═O)—CH═CH—R⁵; R²═H, C₁₋₆alkyl,—CHR⁴—CH₂—COOH or —CHR⁴—CH₂—COOR⁶; and R³-R⁶ are independently alkyl. 9.The method according to claim 6, wherein said rhamnolipids areα-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate,α-L-rhamnosyl-(1,2)-α-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate,or the combination thereof.
 10. The method according to claim 6, whichis in a form suitable for subcutaneous, percutaneous, or intracutaneousadministration. 11-15. (canceled)
 16. A method for treating areas ofcellulite in a human subject, comprising the steps of: identifying ahuman subject suffering from cellulite, and administering into or aroundthe areas of cellulite an effective amount of rhamnolipids, whereby theappearance of cellulite is improved
 17. The method according to claim16, wherein said rhamnolipids are compounds of Formula (I),

wherein: R¹═H, unsubstituted α-L-rhamnopyranosyl, or α-L-rhamnopyranosylsubstituted at the 2 position with —O—C(═O)—CH═CH—R⁵; R²═H, C₁₋₆alkyl,—CHR⁴—CH₂—COOH or —CHR⁴—CH₂—COOR⁶; and R³-R⁶ are independently alkyl.18. The method according to claim 16, wherein said rhamnolipids areα-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate,α-L-rhamnosyl-(1,2)-α-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate,or the combination thereof.
 19. The method according to claim 16, whichis in a form suitable for subcutaneous, percutaneous, or intracutaneousadministration.